Synthesis and Pharmacological Evaluation of Novel Arginine Analogs as Potential Inhibitors of Acetylcholine-Induced Relaxation in Rat Thoracic Aortic Rings

Article abstract of DOI:10.1111/j.1747-0285.2011.01286.x

It is widely appreciated that the vascular endothelium is capable of modulating vascular smooth muscle tone suiting it well for its role as an important regulator of a number of diverse biological processes. Endothelial dysfunction is an early manifestation of atherothrombosis and a consequence of the established disease. Although several arginine derivatives alkylated at one of the guanidino nitrogen were found to inhibit vasorelaxation induced by acetylcholine, activity of the corresponding arginine esters is not reported. The present work was therefore designed to synthesize and evaluate series of novel arginine derivatives to obtain further insight into structure-activity relationship in this series of compounds. Present study involves assessment of activity of these novel compounds on the vascular tone of rat thoracic aorta in comparison with l-arginine analog, that is, l-nitro-arginine methyl ester (l-NAME). Results from the present study showed that full reversal of phenylephrine-mediated contraction was achieved by cumulative applications of acetylcholine (3nm-300μm), which were abolished when the aortic rings were pretreated with l-NAME (300μm). Results from the present study demonstrated that these novel arginine derivatives cause significant yet reversible reduction in acetylcholine-mediated relaxation, similar to that of l-NAME.

Full text of DOI:10.1111/j.1747-0285.2011.01286.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01286.x
Chem Biol Drug Des 2012; 79: 459–469
Research Article
Synthesis and Pharmacological Evaluation of
Novel Arginine Analogs as Potential Inhibitors of
Acetylcholine-Induced Relaxation in Rat Thoracic
Aortic Rings
Manish Jain1, Manoj Kumar Barthwal1,
Wahajul Haq2,*, Seturam B. Katti2 and
Madhu Dikshit1,*
Acetylcholine (ACh) is the gold-standard stimulator of endothelial
cells, particularly in vitro, where it is used extensively to demon-
strate endothelium-dependent relaxation (1,2). Acetylcholine recep-
tors are expressed on endothelial cells of blood vessels, and their
1Division of Pharmacology, Central Drug Research Institute (Council
of Scientific and Industrial Research), 1 M.G. Marg, Lucknow
226001, Uttar Pradesh, India
activation results in elevated endothelial intracellular calcium and
generation of endothelium-derived relaxing factors, which induce
relaxation of the adjacent vascular smooth muscle (Figure 1). It has
been demonstrated that relaxation induced by ACh in isolated tis-
sue is mediated by the release of nitric oxide (NO) (3–6). The
source of endothelial NO appears to be the terminal guanidino
nitrogens of L-arginine. L-Arginine, a guanidino group–containing
basic amino acid, is involved in many important physiological and
pathophysiological processes. The normal intracellular concentration
of L-arginine in the endothelial cell has been reported to be as high
as 800 lM (7), whereas the enzyme's Km for substrate is in the
micromolar range (2.9 lM) and more than enough to saturate NOS.
In vitro studies of Km of L-arginine for NOS suggest that production
of NO is not substrate limited (8). Both acute and chronic adminis-
tration of L-arginine evokes benefits in improving endothelial func-
tion. Endogenous analog of L-arginine, asymmetric dimethylarginine
(ADMA), exerts biological activity by competitively inhibiting NOS
activity. A systemic infusion of ADMA in healthy humans causes
temporary reduction in the cardiac output and induces a reduction
in effective renal plasma flow. Elevations in plasma ADMA have a
high prevalence in hypercholesterolemia, hyperhomocysteinemia,
hypertension, diabetes mellitus, peripheral arterial disease, coronary
artery disease, preeclampsia, and chronic heart failure in which cir-
culating L-arginine concentration has been found to be in the nor-
mal range of 40–100 lM resulting in diminished NOS activity.
Taken together, the above-mentioned results provide a lot of evi-
dence that extracellular ADMA and L-arginine concentrations could
modulate NOS activity (8). Experiments with structural analogs of L-
Arg, for example, L-canavanine, NG-monomethyl-L-Arg, and succinyl-
L-Arg revealed that endothelium-dependent relaxation was inhibited
in the presence of these compounds (9). Other reports suggest that
endothelium-dependent responses induced by ACh and bradykinin in
vivo as well as in vitro were virtually abolished in the presence of
arginine analog, that is, L-NAME (10) implying that NO synthesis
and release accounts for the biological effects of these agents.
2Medicinal and Process Chemistry Division, Central Drug Research
Institute (Council of Scientific and Industrial Research), 1 M.G.
Marg, Lucknow 226001, Uttar Pradesh, India
*Corresponding authors: Madhu Dikshit, madhu_dikshit@cdri.res.in;
Wahajul Haq, w_haq@cdri.res.in
It is widely appreciated that the vascular endothe-
lium is capable of modulating vascular smooth
muscle tone suiting it well for its role as an impor-
tant regulator of a number of diverse biological
processes. Endothelial dysfunction is an early man-
ifestation of atherothrombosis and a consequence
of the established disease. Although several argi-
nine derivatives alkylated at one of the guanidino
nitrogen were found to inhibit vasorelaxation
induced by acetylcholine, activity of the corre-
sponding arginine esters is not reported. The pres-
ent work was therefore designed to synthesize and
evaluate series of novel arginine derivatives to
obtain further insight into structure–activity rela-
tionship in this series of compounds. Present study
involves assessment of activity of these novel com-
pounds on the vascular tone of rat thoracic aorta in
comparison with L-arginine analog, that is, L-nitro-
arginine methyl ester (L-NAME). Results from the
present study showed that full reversal of phenyl-
ephrine-mediated contraction was achieved by
cumulative applications of acetylcholine (3 nM–
300 lM), which were abolished when the aortic
rings were pretreated with L-NAME (300 lM).
Results from the present study demonstrated that
these novel arginine derivatives cause significant
yet reversible reduction in acetylcholine-mediated
relaxation, similar to that of L-NAME.
Key words: arginine analogs, guanido scaffold, rat, thoracic aorta,
vasorelaxation
Mimetics of the natural substrate L-arginine (11) as well as its ana-
logs such as L-NAME and L-iminoethyl-L-ornithine are being increas-
Received 25 February 2011, revised 25 November 2011 and accepted
for publication 26 November 2011
459

Jain et al.
moiety incorporation in the receptor antagonists is often associated
with low selectivity and poor bioavailability after oral administration
(17), which stimulated synthesis of many new, stable, and structur-
ally defined arginine analogs.
There are several methods for the measurements of NO synthase
activity based on various principles. However, there are certain limi-
tations of these assays, for example, citrulline conversion assay
cannot be applied to complex systems that contain relatively low
amounts of NOS (18). Indirect measurements of nitrate and nitrite
cannot resolve whether NO is the primary product of NOS (19). In
context of hemoglobin assay, samples such as the spleen has high
hemoglobin concentration, which diminishes the signal-to-noise
ratio of the assay and may completely obscure enzyme activity (20).
Biological techniques are now widely accepted for different type of
estimations that cannot be easily made by direct chemical or physi-
cal analysis. Indeed biological techniques are preferred over chemi-
cal methods if the final result is required in terms of the biological
potency of the complex material. Thus, in the present study, iso-
lated arterial preparation was used to assess whether the com-
pounds possess any functional inhibitory activity. Isolated arterial
preparation is a very sensitive and reproducible functional bioassay
that helps in direct investigation of NO-dependent vasodilation by
assessing ACh-induced relaxation responses, thus evaluating func-
tional role of NO in hypertension and associated pathologies. Also
this assay is used for in vitro evaluation of the effect of drugs at
modulating smooth muscle tone in normal or pathological condi-
tions. This assay also allows the determination and comparison of
potency (EC50) and efficiency (Emax) of drugs by performing concen-
tration response curves.
Figure 1: Synthesis of nitric oxide in the endothelium of the
vasculature: The endothelial cell on activation by acetylcholine
(ACh) increases intracellular Ca2+ levels, which leads to the activa-
tion of eNOS. L-arginine is metabolized to L-citrulline and NO by
NOS, mainly localized in the cytosol. Formation of NO was inhibited
by arginine analogs, thus prevented ACh-mediated relaxation. This
figure represents effects of NO and NOS inhibitors in various
pathologies.
ingly used as probes to investigate the possible physiological and
pathological roles of NO in hypertension, atherosclerosis, pulmonary
fibrosis, asthma, diabetes, and for inflammatory or infectious dis-
eases such as polyarthritis and septic shock (12). One may likewise
consider using such compounds for similar pathological symptoms
with better potency and enhanced therapeutic index. However, the
wide range of processes in which NO has been implicated, and the
fact that increases or decreases in NO levels might be therapeuti-
cally desirable depending on the condition or even at different
stages of the same condition, pose considerable challenges for drug
development. There can be little doubt that NOS activity is of con-
siderable importance physiologically or that overproduction of NO or
increased NOS activity has the potential to cause harm. Increased
concentration of NO or its stable end products, nitrite ⁄ nitrate, or
the presence of nitrotyrosine, the cellular marker of damaging free
radical, peroxynitrite have prompted clinicians to explore the thera-
peutic potential of NOS inhibitors. Although most interest has
focused on the cardioprotective effects of eNOS, there are some
situations in which eNOS-derived NO might play a proactive role in
disease progression (13,14). Hence, apart from using arginine ana-
logs in understanding NOS-mediated signaling events, they can be
explored in pathological conditions involving excess NOS activity. In
a variety of L-arginine derivatives, terminal guanidine group of the
arginine is modified to methylarginine (15) and nitroarginine (16).
The guanidino group of arginine in many substrates, inhibitors, and
antagonists forms strong ionic interactions with the carboxylate of
aspartic acid moiety, which provides specificity to the basic amino
acid residue in the active site. However, a highly basic guanidino
The structural and biochemical data suggest that the enzyme
through a series of oxidative steps converts guanidino group of
L-arginine to NO. The first step in this sequence is hydroxylation of
one of the guanidino nitrogens followed by further oxidation leading
to the formation of citrulline and NO (21,22). Therefore, major
research effort is focused for designing compounds having guanidino
scaffold or its isostere as NOS inhibitors. An important approach
to obtain more potent inhibitors includes alkylation of one of the
N-e-guanidino group. It is also known from the X-ray crystal struc-
ture of enzyme-bound inhibitors that the active site of NOS is flexi-
ble enough to accommodate molecules different from L-Arg in size,
charge, and hydrophobicity (23). Encouraged by these findings, we
surmised that amino acid analogs having modified guanidino moiety
and dipeptides containing them may lead to better inhibitors. There-
fore, for the present study, we have synthesized derivatives where
the guanido group of arginine has been derivatized to the corre-
sponding tetramethyl guanidine. The tetramethylation of guanido
group does not have any hydrogen of the guanido function and
completely abrogates the possibility of citrulline and NO formation,
but the basicity remains intact for the binding with the carboxylate
of an aspartic acid moiety. In some derivatives, the a-amino group
of ornithine was converted to tetramethyl guanido to explore the
positional specificity of guanido moiety. Thus, a-tetramethylguanyl
ornithine and lysine (compounds 1 and 5) were prepared. We have
also prepared homoarginine methyl ester (2) to see the specificity
of the enzyme as homolog of arginine. In addition to that three di-
peptides with alanine were also synthesized (compounds 6, 7, and
460
Chem Biol Drug Des 2012; 79: 459–469

Arginine Analog Inhibit Acetylcholine-Induced Relaxation
8) to study activity of the dipeptides that might also act as prodrug
and modulate the binding affinity to the enzyme.
DRX-300 MHz FT-NMR spectrometers. The chemical shifts are
reported as parts per million (d p.p.m.) taking tetramethylsilane
(TMS) as an internal standard. Mass spectra were obtained on Mi-
cromass Quattro II Spectrometer using Electro spray ionization mass
spectrometry (ESI MS positive mode). All the compounds are
chromatographically homogeneous and are fully characterized by
spectroscopic methods.
In the present study, we designed some arginine analogs and di-
peptides (Figure 2) viz. TmG-Orn-OMe (1), homoArg-OMe (2),
Orn(TmG)-OMe (3), Lys(TmG)-OMe (4), TmG-Lys-OMe (5), Ala-
Orn(TmG)-OMe (6), Ala-Lys(TmG)-OMe (7) and Ala-Lys (NG)-OMe (8)
and evaluated these arginine analogs on ACh-mediated relaxation
using isolated rat aortic rings. This study will help in identifying the
right analog that may be suitable for the preclinical studies and
also help in consolidating the actual role and molecular mechanism
of different arginine analogs in vascular ring preparations.
Synthesis
Orn(Z)-OMe (I)
A solution of Boc-Orn(Z)-OMe (1.9 g, 5 mmol) was treated with
18% HCl ⁄ dioxane (25 mL) for 2 h at room temperature. The solvent
was removed, and the residue was precipitated by the addition of
excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccators over sodium hydroxide
pellets.
Materials and Methods
Material
Acetylcholine bromide, N-nitro-L-arginine methyl ester (L-NAME),
sodium nitroprusside (SNP), and phenylephrine hydrochloride (PE)
were obtained from Sigma Chemical Co., St Louis, MO, USA. All
other chemicals used in the study were obtained from Indian com-
panies (Merck, Mumbai, India; Glaxo, Worli, India; SRL, Mumbai,
India; or Qualigens, Worli, India). HBTU was purchased from Nova-
biochem (Mumbai, India).
TmG-Orn(Z)-OMe (II)
A solution of HCl.Orn(Z)-OMe (0.8 g, 2 mmol) in acetonitrile 10 mL
was reacted with a solution of 1-benzotriazolyl-N,N,N¢,N¢-tetrameth-
yl-uronium-hexafluoro-phosphate (HBTU) (0.76 g, 2 mmol) (25) in tetr-
ahydrofuran (THF) 10 mL at room temperature for 10–12 h, the
solvent was removed after complete reaction as monitored by TLC,
and the residue was extracted in ethyl acetate. The organic layer
was washed with 5% aq. citric acid, brine, 5% aq. sodium bicar-
bonate, and finally with brine. The ethyl acetate layer was dried
over anhydrous sodium sulfate and concentrated to give crude prod-
uct that was purified by silica gel column chromatography to yield
chromatographically homogeneous compound.
Chemistry
Synthesis of compounds was carried out starting from the commer-
cially available amino acid derivatives viz. Boc-Orn(Z)OMe and
Boc-Lys(Z)OMe (24). The 18% HCl ⁄ dioxane (w ⁄ v) was prepared by
passing dry HCl gas in cold dioxane till required HCl is dissolved.
Thin-layer chromatography was performed on readymade silica gel
plates (UV active, k254 nm; Merck). Chromatography was performed
on silica gel (230–400 mesh). The compounds were detected by
heating the plate to 110 ꢀC for 10 min after spraying with ninhydrin
solution or by exposure to iodine vapors. Column chromatography
was performed on flash silica gel G60 (230–400 mesh, ASTM;
Merck). The 1H spectra were obtained with Bruker DPX-200 or
TmG-Orn-OMe (1)
A solution of TmG-Orn(Z)-OMe (0.38 g, 1 mmol) in methanol was
subjected to catalytic hydrogenation in the presence of 10% Pd ⁄ C
at room temperature. Completion of the reaction was monitored by
Figure 2: Structure of the new arginine analogs. TmG-Orn-OMe (1), Homo Arg-OMe (2), Orn (TmG)-OMe (3), Lys (TmG)-OMe (4), TmG-Lys-
OMe (5), Ala-Orn (TmG)-OMe (6), Ala-Lys (TmG)-OMe (7), and Ala-Lys (NG)-OMe (8).
Chem Biol Drug Des 2012; 79: 459–469
461

Jain et al.
TLC, and catalyst was removed by filtration. The filtrate was evapo-
rated under reduced pressure to obtain the desired compound as
colorless gummy matter. H NMR (DMSO-d6, 300 MHz) d: 4.14 (1H,
a-CH), 3.53 (3H, s, CO2CH3), 2.56 (2 H, m, Orn d -CH2), 2.83 (12 H, s,
guanidine CH3) 1.15–1.70 (6 H, Orn b, c, –CH2).
1
m, Orn a-CH), 3.53 (3H, s, CO2CH3), 2.56 (2 H, m, Orn d-CH2), 2.83
(12 H, s, guanidine CH3) 1.15–1.70 (4H, m, Orn b, c, –CH2).
Boc- Lys(TmG)-OMe (VI)
A solution of Boc-Lys-OMe (1.3 g, 5 mmol) in acetonitrile 30 mL was
reacted with a solution of HBTU (1.89 g, 5 mmol) in THF 20 mL at
room temperature for 10–12 h, the solvent was removed after com-
plete reaction as monitored by TLC, and the residue was extracted in
ethyl acetate. The organic layer was washed with brine, 5% aq.
sodium bicarbonate, and finally with brine. The ethyl acetate layer
was dried over anhydrous sodium sulfate and concentrated to give
crude product that was purified by silica gel column chromatography
to yield chromatographically homogeneous compound.
Boc-Lys-OMe (III)
A solution of Boc-Lys(Z)-OMe (3.95 g, 10 mmol) in methanol was
subjected to catalytic hydrogenation in the presence of 10% Pd ⁄ C
at room temperature. Completion of the reaction was monitored by
TLC, and catalyst was removed by filtration. The filtrate was evapo-
rated under reduced pressure to obtain the desired compound as
colorless gummy matter.
Boc-homoArg(NG-di-Boc)-OMe (IV)
Lys(TmG)-OMe (4)
A solution of Boc-Lys-OMe (1.3 g, 5 mmol) in THF (20 mL) and 1,3-
di-Boc-S-methylisothiourea (1.4 g, 5 mmol) was stirred at room tem-
perature for several hours. After complete reaction as monitored by
TLC (disappearance of 1,3-di-Boc-S-methylisothiourea), the solvent
was removed under reduced pressure. The remaining residue was
extracted with CHCl3 (3 · 10 mL), washed with H2O and brine, and
dried. The product was isolated by flash column chromatography on
silica gel (CHCl3 ⁄ MeOH).
Boc-Lys(TmG)-OMe (0.71 g, 2 mmol) was treated with 18%
HCl ⁄ dioxane (15 mL) for 2 h at room temperature. The solvent was
removed, and the residue was precipitated by the addition of
excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccator over sodium hydroxide
pellets. H NMR (DMSO-d6, 300 MHz) d: 3.72 (3H, s, CO2CH3), 3.43
(3H, m, Lys a-CH, Lys ꢀ-CH2), 2.83 (12 H, s, guanidine CH3) 2.12
(1H, br s, NH), 1.41–1.8 (6 H, m. Lys b, c, d–CH2).
1
HomoArg-OMe HCl (2)
HCl.Lys(Z)-OMe (VII)
The Boc-homoArg(NG-di-Boc)-OMe (1.0 g, 2 mmol) was treated with
18% HCl ⁄ dioxane (15 mL) for 2 h at room temperature. The solvent
was removed, and the residue was precipitated by the addition of
excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccators over sodium hydroxide
A solution of Boc-Lys(Z)-OMe (1.98 g, 5 mmol) was treated with
18% HCl ⁄ dioxane (25 mL) for 2 h at room temperature. The solvent
was removed, and the residue was precipitated by the addition of
excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccator over sodium hydroxide
pellets.
1
pellets. H NMR (CDCl3+ DMSO-d6, 300 MHz) d: 7.3 (2H, br s, NH2),
6.33 (1H, br s, NH), 3.72 (3H, s, CO2CH3), 3.43 (3H, m, Lys a-CH, Lys
ꢀ-CH2), 2.12 (1 H, br s, NH), 1.41–1.80 (6 H, m. Lys b, c, d –CH2).
TmG-Lys(Z)-OMe (VIII)
A solution of VII (0.86 g, 2 mmol) in acetonitrile 10 mL was
reacted with a solution of HBTU (0.76 g, 2 mmol) in THF 10 mL at
room temperature for 10–12 h, the solvent was removed after com-
plete reaction as monitored by TLC, and the residue was extracted
in ethyl acetate. The organic layer was washed with 5% aq. citric
acid, brine, 5% aq. sodium bicarbonate and finally with brine. The
ethyl acetate layer was dried over anhydrous sodium sulfate and
concentrated to give crude product which was purified by silica gel
column chromatography to yield chromatographically homogeneous
compound.
Boc-Orn(TmG)-OMe (V)
A solution of Boc-Orn-OMe (2.4 g, 10 mmol) in acetonitrile 50 mL
was reacted with a solution of HBTU (3.79 g, 10 mmol) in THF
50 mL at room temperature for 10–12 h. Completion of the reaction
was monitored by TLC, solvent was removed, and the residue was
extracted in ethyl acetate. The organic layer was washed with
brine, 5% aq. sodium bicarbonate, and finally with brine. The ethyl
acetate layer was dried over anhydrous sodium sulfate and concen-
trated to give crude product that was purified by silica gel column
chromatography to yield chromatographically homogeneous com-
pound.
TmG-Lys-OMe (5)
A solution of TmG-Lys(Z)-OMe (0.39 g, 1 mmol) in methanol was
subjected to catalytic hydrogenation in presence of 10% Pd ⁄ C at
room temperature. Completion of the reaction was monitored by
TLC and catalyst was removed by filtration. The filtrate was evapo-
rated under reduced pressure to obtain the desired compound as
colorless gummy matter. H NMR (DMSO-d6), 300 MHz) d: 4.14 (1H,
m, Lys a-CH), 3.53 (3H, s, CO2CH3), 2.56 (2 H, m, Lys ꢀ-CH2), 2.83
(12 H, s, guanidine CH3) 1.15–1.70 (6 H, Lys b, c, d –CH2).
Orn(TmG)-OMe (3)
The protected arginine derivative Boc-Orn(TmG)-OMe (0.69 g, 2 mmol)
was treated with 18% HCl ⁄ dioxane (15 mL) for 2 h at room tempera-
ture. The solvent was removed, and the residue was precipitated by
the addition of excess of dry ether. The solid was filtered and then
washed with dry ether and dried in vacuum desiccator over sodium
1
1
hydroxide pellets. H NMR (DMSO-d6, 300 MHz) d: 4.14 (1H, m, Orn
462
Chem Biol Drug Des 2012; 79: 459–469

Arginine Analog Inhibit Acetylcholine-Induced Relaxation
Boc-Ala-Orn(Z)-OMe (IX)
2 h at 0 ꢀC and additional 6 h at room temperature. The DCU was
removed filtration and the clear filtrate was concentrated at reduced
pressure. The residue thus obtained was extracted in ethyl acetate
and the organic layer was washed with 5% aq. citric acid, brine, 5%
aq. sodium bicarbonate, and finally with brine. The ethyl acetate layer
was dried over anhydrous sodium sulfate and concentrated to give
crude product that was purified by silica gel column chromatography
to yield chromatographically homogeneous compound.
A solution of Boc-Ala-OH (0.95 g, 5 mmol) and HOBT (0.76 g,
5 mmol) in dry DMF was stirred in an ice bath. A solution of
HCl.Orn(Z)-OMe (1.5 g, 5 mmol) and N-methylmorpholine (0.55 mL,
5.5 mmol)was added to the reaction mixture followed by the addi-
tion of Dicyclohexylcarbodiimide (1.14 g, 5.5 mmol) in dry dichlorom-
ethane (5 mL) under vigorous stirring at 0 ꢀC. The stirring was
continued for 2 h at 0 ꢀC and additional 6 h at room temperature.
The dicyclohexyl urea (DCU) was removed filtration and the clear
filtrate was concentrated at reduced pressure. The residue thus
obtained was extracted in ethyl acetate and the organic layer was
washed with 5% aq. citric acid, brine, 5% aq. sodium bicarbonate
and finally with brine. The ethyl acetate layer was dried over anhy-
drous sodium sulfate and concentrated to give crude product which
was purified by silica gel column chromatography to yield chromato-
graphically homogeneous compound.
Boc-Ala-Lys-OMe (XIII)
A solution Boc-Ala-Lys(Z)-OMe (0.93 g, 2 mmol) in methanol was
subjected to catalytic hydrogenation in the presence of 10% Pd ⁄ C
at room temperature. Completion of the reaction was monitored by
TLC, and catalyst was removed by filtration. The filtrate was evapo-
rated under reduced pressure to obtain the desired compound as
colorless gummy matter.
Boc-Ala-Orn-OMe (X)
A solution Boc-Ala-Orn(Z)-OMe (0.90 g, 2 mmol) in methanol was
subjected to catalytic hydrogenation in presence of 10% Pd ⁄ C at
room temperature. Completion of the reaction was monitored by
TLC and catalyst was removed by filtration. The filtrate was evapo-
rated under reduced pressure to obtain the desired compound as
colorless gummy matter.
Boc-Ala-Lys(TmG)-OMe (XIV)
A solution of Boc-Ala-Lys-OMe (0.33 g, 1 mmol) in acetonitrile 10 mL
was reacted with a solution of HBTU (0.38 g, 1 mmol) in THF 10 mL
at room temperature for 10–12 h, the solvent was removed after
complete reaction as monitored by TLC, and the residue was
extracted in ethyl acetate. The organic layer was washed with, brine,
5% aq. sodium bicarbonate and finally with brine. The ethyl acetate
layer was dried over anhydrous sodium sulfate and concentrated to
give crude product that was purified by silica gel column chromatog-
raphy to yield chromatographically homogeneous compound.
Boc-Ala-Orn(TmG)-OMe (XI)
A solution of Boc-Ala-Orn-OMe (0.32 g, 1 mmol) in acetonitrile 10 mL
was reacted with a solution of HBTU (0.38 g, 1 mmol) in THF 10 mL
at room temperature for 10–12 h, the solvent was removed after
complete reaction as monitored by TLC and the residue was extracted
in ethyl acetate. The organic layer was washed with, brine, 5% aq.
sodium bicarbonate and finally with brine. The ethyl acetate layer
was dried over anhydrous sodium sulfate and concentrated to give
crude product which was purified by silica gel column chromatography
to yield chromatographically homogeneous compound.
Ala-Lys(TmG)-OMe (7)
A solution of Boc-Ala-Lys(TmG)-OMe (0.22 g, 0.5 mmol) was treated
with 18% HCl ⁄ dioxane (5 mL) for 2 h at room temperature. The sol-
vent was removed, and the residue was precipitated by the addition
of excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccator over sodium hydroxide
1
pellets. H NMR (DMSO-d6, 300 MHz) d: 3.72 (3H, s, CO2CH3), 3.43
(4H, m, Ala a-CH, Lys a-CH, Lys ꢀ-CH2), 2.83 (12 H, s, guanidine
CH3) 2.12 (1 H, br s, NH), 1.41–1.80 (6 H, m. Lys b, c, d –CH2),
1.36 (d, 3 H, J = 7.1 Hz), 1.04 (d, 3 H, J = 6.9Hz).
Ala-Orn(TmG)-OMe (6)
A solution of Boc-Ala-Orn(TmG)-OMe (0.21 g, 0.5 mmol) was treated
with 18% HCl ⁄ dioxane (5 mL) for 2 h at room temperature. The sol-
vent was removed and the residue was precipitated by addition of
excess of dry ether. The solid was filtered and then washed with
dry ether and dried in vacuum desiccator over sodium hydroxide
Boc-Ala-Lys(NG-di-Boc)-OMe (XV)
A solution of Boc-Ala-Lys-OMe (1.65 g, 5 mmol) in THF (20 mL) and
1,3-di-Boc-S-methylisothiourea (1.4 g, 5 mmol) was stirred at room
temperature for 8 h. After complete reaction as monitored by TLC,
the solvent was removed under reduced pressure. The remaining
residue was extracted with CHCl3 (3 · 20 mL), washed with H2O
and brine, and dried. The product was isolated by flash column
chromatography on silica gel (CHCl3 ⁄ MeOH).
1
pellets. H NMR (DMSO-d6, 300 MHz) d: 3.72 (3H, s, CO2CH3), 3.43
(4H, m, Ala a-CH, Orn a-CH, Orn d-CH2), 2.83 (12 H, s, guanidine
CH3) 2.12 (1 H, br s, NH), 1.41–1.80 (4H, m. Orn b, c, –CH2), 1.36
(d, 3 H, J = 7.1 Hz), 1.04 (d, 3 H, J = 6.9Hz).
Boc-Ala-Lys(Z)-OMe (XII)
A solution of Boc-Ala-OH (0.95 g, 5 mmol) and HOBT (076 g, 5 mmol)
in dry DMF was stirred in an ice bath. A solution of HCl.Lys(Z)-OMe
(1.6 g, 5 mmol) and N-methylmorpholine (0.55 mL, 5.5 mmol) was
added to the reaction mixture followed by the addition of Dic-
yclohexylcarbodiimide (1.14 g, 5.5 mmol) in dry dichloromethane
(5 mL) under vigorous stirring at 0 ꢀC. The stirring was continued for
Ala-Lys (NG)-OMe (8)
A solution of Boc-Ala-Lys (NG-di-Boc)-OMe (0.58 g, 1.0 mmol) was
treated with 18% HCl ⁄ dioxane (10 mL) for 2 h at room temperature.
The solvent was removed, and the residue was precipitated by the
addition of excess of dry ether. The solid was filtered and then
Chem Biol Drug Des 2012; 79: 459–469
463

Jain et al.
washed with dry ether and dried in vacuum desiccator over sodium
hydroxide pellets. H NMR (DMSO-d6, 300 MHz) d 7.3(2H, br s, NH2),
6.33 (1H, br s, NH), 3.72 (3H, s, CO2CH3), 3.43 (4H, m, Ala a-CH, Lys
a-CH, Lys ꢀ-CH2), 2.12 (1 H, br s, NH), 1.41–1.80 (6 H, m. Lys b, c, d
–CH2), 1.36 (d, 3 H, J = 7.1 Hz), 1.04 (d, 3 H, J = 6.9 Hz).
of ACh in the presence of various test agents in the study was
depicted as a percentage of the control. Percent relaxation was
given by 100· [agonist-induced relaxation in the absence or pres-
ence of drugs ⁄ PE (1 lM)-induced contraction]. Relaxation responses
to ACh (3 nM–300 lM) were obtained to establish the integrity of
the endothelium. Cumulative concentration response curves of ACh
were fitted by nonlinear regression using in-house software, and
the EC50 values of the rings to ACh were calculated from the lin-
ear range of this concentration response curve (34). Rings with
functional endothelium exhibited significant (>50%) relaxations,
whereas endothelium-denuded rings lacked significant relaxation to
ACh (6,33).
1
Effect of arginine analogs on ACh-induced
relaxations
Animals. Ten-week-old Sprague-Dawley rats (100–120 g) obtained
from the National Laboratory Animal Centre of Central Drug
Research Institute, Lucknow, were used in the present study. All
the procedures involving Sprague-Dawley rats were subject to Insti-
tutional Animal Ethical Committee (IAEC) guidelines. The animals
were housed in polypropylene cages and maintained on standard
chow diet and water ad libitum and on 12-h ⁄ 12-h light ⁄ dark cycle
at temperature 25€2 ꢀC, humidity 45–55%, and ventilation 10–
12 exchange ⁄ h.
Rings were then pre-incubated with test compounds numbered 1–8
or L-NAME at 300 lM concentration for 30 min (35). Cumulative
concentration-dependent response to ACh (3 nM–300 lM) were sub-
sequently obtained in PE (1 lM) precontracted rings. In another set
of experiments, NO donor (SNP, 1 nM–1 lM)-induced relaxation was
monitored in the absence or presence of 2, 7, or L-NAME. Rings
were subsequently washed and allowed to rest for at least 30 min,
and subsequently, PE (1 lM)-induced contraction and ACh (3 nM–
300 lM)-induced relaxation were evaluated to assess the recovery
after washing off the test substances.
Rat aortic ring preparation. Rats were euthanized in accordance
with the institution guidelines under an approved protocol. Chest
was opened, and the thoracic aorta was excised and placed in ice-
cold Kreb's bicarbonate solution (composition: NaCl 113 mM; KCl
5 mM; CaCl2 1.4 mM; MgSO4 0.9 mM; NaHPO4 1.2 mM; NaHCO3
25 mM; Glucose 11.5 mM; pH 7.4) (26,27) and trimmed of adhering
connective tissue and fat. Transverse 4-mm-wide rings were cut
and mounted in 10 mL organ baths containing Kreb's solution,
maintained at 37 ꢀC and continuously oxygenated with 95% O2 to
5% CO2. The rings were equilibrated for 90 min, during which the
bathing fluid was changed every 15 min and the tissue was kept
under a constant tension of 2 g throughout the experiment (28,29).
Isometric measurements were recorded with force transducers
(FSG-01, Budapest, Hungary) using S.P.E.L. solution Pack for Experi-
mental Laboratories ADVANCE ISOSYS data acquisition and analysis
software (Experimetria, Budapest, Hungary) (30). Endothelium from
some of rings from the same animal was removed by gently
rubbing the intimal surface of the aorta with a fine forceps over a
filter paper soaked with Kreb's bicarbonate solution.
Statistical analysis
Each experimental group consisted of a minimum of four aortic
rings from different rats. Values have been expressed as mean € -
SEM. Emax of PE (1 lM) was considered as 100%, and the relaxa-
tion on cumulative additions of ACh in the presence of various test
agents was calculated as percentage of PE-induced contraction. The
EC50 (with 95% CI) of ACh was calculated using probit analysis,
which was based on maximum likelihood iterative procedure. The
method of calculating EC50 is indicated by example in Supporting
Information section (Table S1). The significance of difference
between different groups was compared by one-way analysis of
variance (ANOVA), and for each concentration, the significance of
mean difference between the groups was compared using New-
man–Keuls post hoc test. The difference was considered to be sig-
nificant when the p-value was <0.05.
Assessment of tissue contractility and viability. To assess the max-
imum tissue contractility and viability, the rings were exposed to
80 mM KCl Kreb's solution. The response was measured before and
after the experimental protocol in each aortic ring (31). Based on
the maximal response obtained by 80 mM KCl Kreb's buffer, percent
contraction of the each aortic ring was calculated in each experi-
ment. Data were expressed as increase in tension calculated as a
percentage of the KCl-induced contraction and were plotted against
log agonist concentration. The percentage contraction was given by
100· (agonist-induced contraction in the absence or presence of
drugs ⁄ 80 mM KCl-induced contraction) (32).
Results
Synthesis of test compounds
The test compounds were synthesized as aforementioned, and a
summary of the important data has been presented in Table 1.
Effect of L-Arginine analogs and L-NAME on
ACh-induced relaxations
Endothelium-intact rat aortic rings contracted with phenylephrine
(EC80, 1 · 10)6 M) produced stable contractions (0.6 € 0.02 g). Ace-
tylcholine (3 nM–300 lM) produced concentration-dependent relax-
ations in PE precontracted rings. Incubation of a variety of novel
arginine analogs or dipeptides altered the vascular tone of rat aor-
tic rings (Figure 3). Incubation of the aortic rings for 30 min with
L-NAME, 2, 6, 7, or 8 (300 lM) augmented basal tone of the rings
Experimental protocol
After the assessment of maximum tissue contractility, the aortic
rings were contracted with submaximal concentration of phenyl-
ephrine (1 lM, i.e., EC80) (6,33), obtained from concentration
response curve to phenylephrine (10)10–10)3 M). Emax of PE was
considered as 100%, and the relaxation on cumulative additions
464
Chem Biol Drug Des 2012; 79: 459–469

Arginine Analog Inhibit Acetylcholine-Induced Relaxation
Table 1: Isolated yields and mass spectroscopic results of new
compounds
Table 2: Effect of test compounds and L-NAME on acetylcholine
(ACh)-induced relaxation
ESMS M ⁄ Z [M+H]+
Treatment
EC50 of ACh (lM)
95% CI
Compound no. Compound structure Yield (%) Observed Calculated
Control (n = 21)
L-NAME (n = 12)
300 lM
200 lM
100 lM
50 lM
Compound 1 (n = 6)
300 lM
Compound 2 (n = 4)
300 lM
200 lM
0.105
0.052–0.22)
1
2
3
4
5
6
7
8
TmG-Orn-OMe
91
85
90
88
90
80
82
91
245
203
245
259.3
259
316
330
274
245
203
245
259
259
316
330
274
8416.3*
1 050 328*
645 629*
225.4–>13 240
55 197–1 998 6160
72 149–>5 777 427
210.1–3322.4
HomoArg-OMe
Orn(TmG)-OMe
Lys(TmG)-OMe
835.5*
TmG-Lys-OMe
Ala-Orn(TmG)-OMe
Ala-Lys(TmG)-OMe
Ala-Lys (NG)-OMe
3.85*#
1.40–10.5
2650.7*#
472.7*#
75.82*#
3.21*#
183.4–3806
146.8–1521.7
14.3–399.3
1.7–5.8
100 lM
50 lM
Compound 3 (n = 6)
300 lM
Compound 4 (n = 6)
300 lM
Compound 5 (n = 6)
300 lM
Compound 6 (n = 6)
300 lM
5.14*#
7.78*#
1.76*#
7.94*#
2.03–13.0
3.34–18.1
0.83–3.7
2.98–21.1
Compound 7 (n = 6)
300 lM
200 lM
100 lM
50 lM
32813.6*#
28.93*#
0.76*#
122.3–41 362
9.2–90.5
0.19–2.98
0.12–0.92
0.33*#
Compound 8 (n = 6)
300 lM
0.214#
0.919–0.49
Figure 3: Bar diagram representing effect of test compounds
(300 lM) or L-NAME (300 lM) on the basal tone after 30-min incu-
bation with endothelium-intact rat aortic rings. Results have been
represented as % contraction in relation to maximum contraction
produced by 80 mM KCl. *p < 0.05, **p < 0.01, and ***p < 0.001
6, 7, 8 versus L-NAME.
Comparison of the EC50 values of ACh (3 nM–300 lM) obtained in the cumu-
lative concentration-dependent responses in phenylephrine (1 lM) precon-
tracted endothelium-intact rat aortic rings after incubation with L-NAME or
1, 2, 3, 4, 5, 6, 7, 8. *p < 0.001 versus control, #p < 0.001 versus L-NAME
(300 lM).
(Figure 3), while at lower doses, these test substances had no
effect. Compound 1, 3, 4, or 5 did not exhibit any significant effect
at 300 lM concentrations on the basal tone or PE-induced contrac-
tions in the aortic rings.
5, 6, and 8 also exhibited reduction in the ACh-induced relaxation
at 300 lM, but their effect was less than L-NAME, 2 and 7
(Table 2, Figure 5). Effect of 1, 3, 4, 5, 6, and 8 was, thus, not
evaluated at lower concentrations on ACh-induced relaxations.
Sodium nitroprusside induced concentration-dependent relaxation in
the PE (1 lM) precontracted endothelium-intact rings. Maximal
relaxation evoked by SNP (1 lM) was 116 € 6. Sodium nitroprus-
side-induced relaxations were almost similar in rings pre-incubated
with L-NAME (115 € 6), 2 (107.3 € 2.0) or 7 (104.1 € 3.2). The
results obtained indicate that among the compounds tested, com-
pound 7 was most efficacious and 8 was least effective as
assessed by the EC50 concentration of ACh required to relax the
aortic rings (Table 2) in the presence or absence of these com-
Acetylcholine-induced concentration-dependent relaxations in the
endothelium-intact rings were evaluated in the presence of
L-NAME or test compounds at 300 lM concentration. Maximal
relaxation induced by the ACh was significantly reduced in the
presence of L-NAME (36.77 € 4.05) as compared to control rings
(85.74 € 2.06), (Table 2, Figure 4). Results obtained regarding
reduction in ACh-mediated relaxation in the presence of L-NAME
was similar to earlier reports. L-NAME (300 lM) has previously
been shown to inhibit vasorelaxation evoked by ACh by upto 80%.
Similarly, L-NAME at 300 lM concentration has been previously
shown to increase the basal tone (35,36). Similar results were
obtained with compound 2 or 7 (Figure 4). Inhibitory effect of
L-NAME, 2, or 7 was almost comparable at 300 lM; therefore,
their effect was also studied at lower doses (50, 100, and
200 lM) on ACh-induced relaxations. Effect of L-NAME was, how-
ever, more in comparison with the compounds 2 and 7 (50, 100
and 200 lM) (Table 2, Figure S1). Other test compounds 1, 3, 4,
pounds.
Rank
order
of
the
test
compound
was
8 < 5 < 1 < 3 < 4 < 6 < 2 < 7 (Table 2). Compound 2 or 7 did
not showed any effect on basal tone in endothelium-denuded
rings. Complete recovery was observed in the PE-induced contrac-
tions and ACh-induced relaxations following washing of the test
agent treated rings, indicating that the effect of compounds was
reversible in nature. KCl responses were also completely restored
after washing off the compounds, indicating no adverse effect on
the vascular tissue by any of these compounds.
Chem Biol Drug Des 2012; 79: 459–469
465

Jain et al.
A
B
Figure 4: Acetylcholine (3 nM–300 lM)-induced concentration-dependent relaxation in the phenylephrine hydrochloride (1 lM) precontract-
ed rat aortic rings in the presence of L-NAME (300 lM) (n = 12), 2 (300 lM) (A), 7 (300 lM) (B) (n = 3), or vehicle (control) (n = 21). Results
have been represented as mean € SEM. *p < 0.01 and **p < 0.001 L-NAME, 2, or 7 versus control.
A
B
C
D
E
Figure 5: Acetylcholine (3 nM–300 lM)-induced concentration-dependent relaxation in the phenylephrine hydrochloride (1 lM) precontract-
ed rat aortic rings in the presence of L-NAME (300 lM) (n = 12), 1 (A), 3 (B), 4 (C), 5 (D), 6(E) (300 lM) (n = 3), or vehicle (control) (n = 21).
Results have been represented as mean € SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 L-NAME, 1, 3, 4, 5, or 6 versus control. $p < 0.05,
$$p < 0.01 1, 3, 4, or 6 versus L-NAME.
identified, which altered the vascular tone. The mechanism of
action of Nx-substituted arginine analogs such as L-NAME is to
compete with L-arginine for binding to the active site of NOS (37).
Similarly, several arginine derivatives alkylated at one of the guani-
dino nitrogens also exhibited potent activity. In the present study,
we have synthesized a series of compounds comprising arginine
methyl esters and N-G tetramethylated methyl esters to obtain fur-
ther insight into the structure–activity relationship in this series of
compounds. Furthermore, we also synthesized corresponding hom-
oarginine derivatives and their dipeptide derivatives 1–8 (Figure 1).
The compounds exhibited inhibition of ACh-induced relaxations
(Table 2).
Discussion
The present study explored the pharmacological properties of novel
arginine methyl esters and N-G tetramethylated methyl esters as
well as their corresponding homoarginine and their dipeptide deriv-
atives. Eight L-arginine analogs were investigated to elucidate their
effect on vasoreactivity in the endothelium-intact rat aortic rings.
Results from the current studies showed that using an in vitro bio-
assay system, of rat aortic ring segments with these arginine ana-
logs or dipeptide derivatives, attenuation in ACh-induced
vasorelaxation was observed. Test compounds were evaluated and
compared with commercially available arginine analog L-NAME, for
their ability to inhibit ACh-induced relaxation.
The dipeptide analog Ala-Lys (TmG)-OMe (7) was most active and
was equipotent as compared to L-NAME in reducing the relaxant
response to ACh in rat aortic rings. Based on our results, it can be
proposed that some of the compounds tested exhibited similar
activity as that of L-NAME at 300 lM concentration in inhibiting
During the last two decades, considerable research efforts have
been expanded in developing small organic molecules as L-arginine
derivatives. In this endeavor, most important prototypes have been
L-arginine derivatives, namely L-nitro-arginine and L-NAME were
466
Chem Biol Drug Des 2012; 79: 459–469

Arginine Analog Inhibit Acetylcholine-Induced Relaxation
ACh-mediated relaxation of rat aortic rings. However, the N-G
tetramethylated derivatives of L-arginine were generally not very
active in the present assay system. Compound 2 has shown signifi-
cant activity (Table 2, Figure 3) emphasizing homo arginine methyl
ester as NOS inhibitor. Both compounds 2 and 7 have shown com-
parable inhibition in ACh-mediated relaxation vis-a-vis L-NAME
experiments. However, basal tension was changed by compound 2
but not by compound 7. Structurally the compounds are different
which may be one of the reasons for showing different contractile
responses in basal tone. It is well known that endothelial cells gen-
erate significant amounts of reactive oxygen species (ROS) (38);
hence, it is conceivable that endogenously produced ROS might also
regulate vascular tone. While there is every possibility that com-
pound 2 in addition to its NOS inhibitory property may have ROS
scavenging effect, we feel that it is a remote possibility considering
the nature of these compounds as L-arginine analogs and not ROS
scavengers. However, the consideration of other possible contractile
mechanisms like Ca2+-dependent and Ca2+-independent pathways
cannot be ruled out for explaining the different intensity of contrac-
tile responses with compounds 2 and 7.
demonstrated inhibition of ACh-mediated relaxation in the endothe-
lium-intact rat aortic rings. The ability of the synthetic derivatives 1–8
to inhibit ACh-induced vasorelaxation exhibited mixed trends, where
derivatives 7 and 2 corresponding to homoarginine were quite inter-
esting. The inhibitory effect of compounds 7 and 2 on ACh-induced
relaxations was significant, and compound 7 was almost equipotent
to L-NAME. The potent inhibitory activity of compound 2 indicated
that the compound viz. homoarginine methyl ester with free guanido
groups also act as inhibitor and the effect was regioselective. The role
of a proton on guanido group seemed to be crucial as tetra-alkylated
guanido derivatives 1, 3, 4, and 5 could not show activity comparable
to L-NAME. The study, thus, opens new avenues for design and devel-
opment of potent arginine analogs, which may serve an important tool
for the understanding and treatment of related diseases.
Acknowledgments
Financial support from Central Drug Research Institute and
NWP0034 is acknowledged. Fellowship from ICMR to MJ is also
acknowledged. We are also grateful to Mr MPS Negi from Biometry
and statistics division, CDRI, Lucknow, for helping in the statistical
analysis of the data. The spectroscopic data provided by SAIF, CDRI,
Lucknow, are also acknowledged.
It may be noted that the arginine derivatives are stereoselective in
their action as the D-NAME enantiomer of L-NAME has been
reported to be inactive toward NOS inhibition (39). To explore the
effect of regioselectivity, homo arginine methyl ester was prepared
and tested for inhibitory potential of ACh-induced relaxation. The
homo arginine derivative (2) with free guanido group was expected
to behave as substrate for NOS, but it has shown significant inhibi-
tory activity against ACh-induced relaxation, clearly indicating that
the arginine derivatives were also regioselective. The compound 2
apparently able to bound to the active site because of free guanido
group but failed to trigger NO generation. Similar trend was also
observed with dimethyl guanido substituted derivatives where the
homoarginine derivatives have shown better activity as compared to
arginine derivatives (Table 2). We have observed another interesting
feature that the dipeptide derivatives with dimethyl guanido group
have show better activity as compared to corresponding monomers.
The dipeptide derivatives (6 and 7) have shown better activity as
compared to monomers (3 and 4). Higher activity of the dipeptide
derivatives might be attributed to the presence of additional basic
amino group that may facilitates the binding of the ligands to the
acidic active site of the enzyme. Among all the compounds, the
dipeptide derivative (7) has shown highest activity. This further
strengthens the earlier observation that the enzyme active site is
flexible and can accommodate molecules bigger than arginine. To
ascertain the mechanism of action of these compounds, involving
inhibition of endothelial NOS, we further evaluated the two most
active compounds 2 and 7 against SNP-mediated relaxation.
Sodium nitroprusside-induced relaxations were not affected by L-
NAME, 2 or 7. The results obtained, thus, suggest that the com-
pounds seem to inhibit endothelial NOS, which seem to account for
their effect against ACh-induced relaxations in the rat aortic rings.
Conflict of Interest
The authors declare that there are no conflicts of interest.
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Chem Biol Drug Des 2012; 79: 459–469

Arginine Analog Inhibit Acetylcholine-Induced Relaxation
Table S1. Method of calculating EC50 value by example of control
Supporting Information
responses.
Additional Supporting Information may be found in the online ver-
sion of this article:
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Figure S1. Acetylcholine (3 nM–300 lM) induced concentration-
dependent relaxation in the PE (1 lM) precontracted rat aortic rings
in the presence of 2, 7 (50, 100, 200, and 300 lM), or vehicle (con-
trol).
Chem Biol Drug Des 2012; 79: 459–469
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